non-destructive dna extraction methods that yield dna

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Non-destructive DNA extraction methods that yield DNA barcodes Non-destructive DNA extraction methods that yield DNA barcodes

in spiders in spiders

Reva Ann Bork University of Northern Iowa

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i

NON-DESTRUCTIVE DNA EXTRACTION METHODS THAT YIELD DNA BARCODES IN

SPIDERS

A Thesis Submitted

in Partial Fulfillment

of the Requirements for the Designation

University Honors

University of Northern Iowa

Reva Ann Bork

May 2015

NON-DESTRUCTIVE DNA EXTRACTION METHODS ii

This Study by: Reva Ann Bork

Entitled: Non-destructive DNA extraction methods that yield DNA barcodes in spiders

has been approved as meeting the thesis or project requirement for the Designation

University Honors

________ ______________________________________________________

Date Dr. Theresa Spradling, University of Northern Iowa Biology

________ ______________________________________________________

Date Dr. L. Brian Patrick, Dakota Wesleyan University Biology

________ ______________________________________________________

Date Dr. Jessica Moon, Director, University Honors Program

NON-DESTRUCTIVE DNA EXTRACTION METHODS iii

Acknowledgements

First, I would like to thank Dr. L. Brian Patrick and Dakota Wesleyan University for allowing

me to work with them and use their facilities. Next, I would like to thank the CORE Sequencing

Facility at Black Hills State University and Oxana Gorbatenko, who performed all the DNA

concentration and PCR work. Lastly, I would like to thank Dr. Theresa Spradling, who worked

as my thesis advisor even though I did not do the research with her.

NON-DESTRUCTIVE DNA EXTRACTION METHODS iv

Abstract

DNA barcodes are short sequences of nucleotides that differ from species to species (Hebert et

al., 2003; Hebert & Gregory, 2005). DNA barcoding is very important in helping to reconstruct

phylogenetic trees and to confirm the identity of threatened or endangered species in the wild.

Additionally, it has become increasingly popular in the food industry to test the accuracy of the

food being sold in fast food chains (Wong and Hanner, 2008). For scientific purposes, non-

destructive DNA extraction techniques need to be explored because they allow for preservation

of the voucher specimen, which is particularly important for rare or extinct museum specimens.

Using a commercial DNA extraction kit could make the process of extracting DNA from small

voucher specimens more regulated, and thus more universal. It is already known that DNA can

be extracted from spiders in a non-destructive manner. This study addressed whether or not DNA

can be extracted from small spiders (1-3 millimeters in size) in a non-destructive manner using a

commercial DNA extraction kit, such as the Qiagen DNeasy Blood and Tissue Kit. From this

research, it was determined that DNA can be extracted in a non-destructive manner using a

commercial kit in fresh collections of spiders, thus allowing DNA barcoding while saving the

specimen as a voucher. However, in preserved voucher specimens, four years or older, non-

destructive DNA extraction methods using a commercial kit were not as effective, preventing

DNA barcoding.

NON-DESTRUCTIVE DNA EXTRACTION METHODS v

Table of Contents

Introduction………………………………………………………………………………………1

Literature Review………………………………………………………………………………..4

What is a DNA Barcode?………………………………………………………………..4

Efficacies of DNA Extraction Techniques……………………………………………...6

Non-destructive DNA Extraction Techniques………………………………………….7

DNA extraction of Spiders to Gain Barcodes…………………………………………..8

Methodology…………………………………………………………………………………….10

Fresh Collections………………………………………………………………………..10

Voucher Collections…………………………………………………………………….10

Photographic Evidence…………………………………………………………………11

DNA Extraction…………………………………………………………………………11

PCR……………………………………………………………………………………...11

Results…………………………………………………………………………………………...13

Fresh Collections………………………………………………………………………..13

Voucher Collections…………………………………………………………………….17

Discussion………………………………………………………………………………………21

Conclusions……………………………………………………………………………………...22

References………………………………………………………………………………………25

NON-DESTRUCTIVE DNA EXTRACTION METHODS 1

Introduction

In a world where there are millions of species, DNA barcoding is one way that current

species can be confirmed and new species can be discovered and logged (Hebert, Cywinska, &

Ball, 2003; Barrett & Hebert, 2005; Hebert & Gregory, 2005; Rowley et al., 2007). DNA

barcodes are short sequences of DNA nucleotides that are unique to a certain species (Hebert et

al., 2003; Hebert & Gregory, 2005). These unique sequences, along with morphological

descriptions, can then be entered into a database to help differentiate species and fill in gaps in

phylogenetic trees (Hebert et al., 2003; Hebert & Gregory, 2005). DNA barcoding works by

extracting mitochondrial DNA and amplifying the cytochrome c oxidase I (COI) gene via

polymerase chain reaction (PCR; Hebert et al., 2003; Barrett & Hebert, 2005; Hajibabaei et al.,

2005; Mitchell, 2008; Wong & Hanner, 2008; Moulton, Song, & Whiting, 2010; Zeale, Butlin,

Baker, Less, & Jones, 2011; Newmaster, Grguric, Shanmughanandhan, Ramalingam, &

Ragupathy, 2013). This gene is ideal to barcode because it is found in all animal species and has

regions of conserved sequence that allow amplification with common primers (Hebert et al.,

2003; Barrett & Hebert, 2005; Hebert & Gregory, 2005; Mitchell, 2008).

It was already known that DNA could be extracted from spiders in a non-destructive

manner. The question to be answered from research was whether or not DNA could be extracted

from small spiders (1-3 millimeters in size) in a non-destructive manner using a commercial

DNA extraction kit, such as the Qiagen DNeasy Blood and Tissue Kit.

In the realm of spiders, DNA barcoding had the potential to be a more accurate way to

identify what species a smaller organism belongs to (Rowley et al., 2007). Ideal candidates for

this experiment were small in size (1-3 millimeters in size), and were not good candidates to use

a leg for DNA extraction because of the small amount of DNA contained in their legs. Not


destroying these small organisms was important because many of them were voucher species

that must be kept (Rowley et al., 2007).

The purpose of this quantitative research was to extract sequence-able DNA, known as a

DNA barcode, from small spiders using non-destructive DNA extraction techniques and the

Qiagen DNeasy kit (Qiagen, 2006). The DNA extracted from these spiders could be entered into

a national database, such as GenBank or the Consortium for the Barcode of Life (CBOL), in

order to identify existing species and confirm new species. Non-destructive DNA extraction

techniques needed to be explored because they allow for preservation of the voucher specimen.

In addition, rare or extinct museum specimens are ideal candidates for non-destructive DNA

extraction techniques because these techniques do not harm the specimen. Additionally, using a

commercial DNA extraction kit could make the process of extracting DNA from small voucher

specimens more regulated, and thus more universal.

The significance of this study is that it would allow non-destructive DNA extraction

techniques to become more universal. This would be especially important in logging DNA

barcodes of museum specimens or voucher collections into GenBank or CBOL. With a more

universal non-destructive DNA extraction method, DNA could easily be extracted from museum

specimens that have gone extinct and voucher specimens that should not be harmed.

Additionally, a more streamlined DNA extraction technique could allow small labs to extract

DNA from several hundred-thousand samples a year, which would result in a more complete

phylogenetic tree.

In looking at the research that has already been done on this topic, four distinct areas of

research became apparent: what DNA barcodes are, efficacies of DNA extraction techniques,

non-destructive DNA extraction techniques, and DNA extraction of spiders to gain DNA


barcodes. From looking broadly at the topic of non-destructive DNA extraction techniques, it is

observed that little research has been done on whether or not DNA can be extracted using non-

destructive methods and a commercial DNA extraction kit. When the topic is narrowed even

further, it becomes evident that there is little existing research on the use of non-destructive DNA

extraction techniques using a commercial kit in spiders.


Literature Review

What is a DNA Barcode?

In recent years, the importance of DNA barcoding has increased because of the need to

make as complete of a phylogenetic tree as possible in order to differentiate species and discover

new species (Hebert et al., 2003; Hebert & Gregory, 2005). DNA barcodes are short sequences

of nucleotides that differ from species to species (Hebert et al., 2003; Hebert & Gregory, 2005).

A DNA barcode can be obtained by extracting DNA from the COI gene of most animals (Hebert

et al., 2003; Hebert & Gregory, 2005). The COI gene is ideal for DNA barcoding because it

provides a greater range of phylogenetic signal, and in order to obtain a DNA barcode, the DNA

sequence must differ enough at genetic markers to show species resolution (Hebert et al., 2003;

Mitchell, 2008; Casiraghi, Labra, Ferri, Galimberti, & De Mattia, 2010; Zeale et al., 2011). In

addition, the COI gene has a high incidence of third-position base substitutions, so it has a higher

rate of molecular evolution than 12s or 16s rDNA (Hebert et al., 2003). Lastly, the COI gene is

ideal for DNA barcoding because there are many copies of the mitochondrial genome in each

cell (Hajibabaei et al., 2005).

DNA barcoding is unique because it is one of the first tests that is accurate enough to

differentiate between many different animals at the species level (Wong & Hanner, 2008). In

fact, it has been estimated that DNA barcoding can provide species-level resolution in 95-97% of

cases (Hebert & Gregory, 2005). DNA barcoding uses a standardized methodology to enter these

“molecular identification tags” into a universal database (Hebert et al., 2003; Hebert & Gregory,

2005; Mitchell, 2008; Wong & Hanner, 2008; Dittrich-Schroder, Wingfield, Klein, & Slippers,

2012). Thus, reference sequences of the same species can be used to ensure that correct species

identifications are made (Mitchell, 2008; Zeale et al., 2011; Dittrich-Schroder et al., 2012).


Furthermore, DNA barcoding and the use of DNA sequencing libraries, such as GenBank and

CBOL, allow for quicker identification and differentiation of new species (Hebert & Gregory,

2005; Mitchell, 2008; Dittrich-Schroder et al., 2012). In contrast, DNA barcoding can also be

used to prevent mislabeling an old species as a new species. Actually, DNA barcoding makes it

easier to identify morphologically different sexes and life stages of the same species and can help

clarify problems of synonymy (Hebert et al., 2003; Hebert & Gregory, 2005; Casiraghi et al.,

2010; Dittrich-Schroder et al., 2012).

DNA barcoding has made taxonomic systems more accessible, which is beneficial to

ecologists, conservationists, and food safety companies (Hebert & Gregory, 2005; Mitchell,

2008). The use of DNA barcoding is not just limited to science. Commercially, DNA barcoding

can be used to detect contamination and substitutions in herbal products (Newmaster et al.,

2013). In recent years, DNA barcoding has been used to detect market substitutions in North

American seafood (Wong & Hanner, 2008). In fact, DNA barcoding was recently used to

confirm that a toxic puffer fish had been smuggled into a Chicago market illegally and under a

false name (Wong & Hanner, 2008). DNA barcoding is ideal for detecting product

contamination and substitution because it is widely accessible as a commercial tool that is fast

and cost effective (Hebert & Gregory, 2005; Wong & Hanner, 2008; Newmaster et al., 2013).

These findings are important because contaminations and substitutions in herbal products and

seafood markets can be a health risk to consumers and may not be approved by the Food and

Drug Administration (FDA; Newmaster et al., 2013). Currently, there are no standards for the

authentication of herbal products (Newmaster et al., 2013). However, the contaminants and

substitutions that DNA barcoding has uncovered may urge the herbal industry to reconsider their


lack of standards. Clearly, DNA barcoding has made an impact in the realm of taxonomy and in

market substitutions commercially.

Efficacies of DNA Extraction Techniques

Since DNA barcoding is fairly new in the field of science, the efficacy of DNA barcoding

and DNA extraction is a big topic. Because DNA barcoding is so standardized, a generalization

of the protocol for DNA barcoding would allow for more identifications to be made (Mitchell,

2008; Casiraghi et al., 2010). As DNA extraction is the critical first step in all DNA barcodes, it

seems obvious that streamlining DNA extraction protocols could help standardize DNA

barcoding (Ivanova, Dewaard, & Hebert, 2006; Lucentini, Caporali, Palomba, Lancioni, &

Panara, 2006).

There are several facets that an ideal DNA extraction protocol should follow. For

instance, an ideal DNA extraction protocol should yield the most DNA possible, limit amounts

of DNA degraded, be cost-effective, and efficient in terms of time and labor (Chen, Rangasamy,

Tan, Wang, & Siegfried, 2010). Additionally, DNA extraction protocols should not use toxic

chemicals or produce hazardous wastes (Chen et al., 2010; Dittrich-Schroder et al., 2012).

Since the aim of DNA extraction is to purify the DNA by binding it to a membrane or by

fractionation (Hajibabaei et al., 2005), a streamlined DNA extraction protocol, such as one that

uses a commercial kit, is more cost-effective and allows for hundreds of thousands of samples to

be barcoded by the same facility each year (Hajibabaei et al., 2005; Wong & Hanner, 2008).

There are several advantages to using a commercial kit. The Qiagen DNeasy Kit is convenient,

efficient, and does not require the use of a fume hood (Chen et al., 2010). The Qiagen DNeasy

kit purifies DNA by binding DNA to the membrane of silica (Hajibabaei et al., 2005, Qiagen,

2006). In general, silica-based approaches are ideal for high-throughput DNA barcoding of small


tissue samples (Hajibabaei et al., 2005). In addition, silica-based approaches, such as the Qiagen

DNeasy Blood and Tissue Kit, have been found to be the best for extracting DNA from archived

specimens (Hajibabaei et al., 2005). Thus, the Qiagen DNeasy kit is appropriate for this project.

Non-destructive DNA Extraction Techniques

There is one problem with the methods used in DNA barcoding; DNA barcoding often

involves destroying the organism from which DNA is being extracted (Phillips & Simon, 1995;

Rholand, Siedel, & Hofreiter, 2004; Rowley et al., 2007; Hunter, Goodall, Walsh, Owen, & Day,

2008; Miller, Beentjes, van Helsdingen, & IJland, 2013). This is problematic because specimens

that are caught are often used as a voucher for a new species (Rowley et al., 2007; Hunter et al.

2008, Casquet, Thebaud, & Gillespie, 2012). Additionally, barcoding an endangered species or

an ancient organism that resides in a museum should be avoided to protect the integrity of the

museum collection (Wasko, Martins, Oliveira, & Foresti, 2003; Rohland et al., 2004; Hajibabaei

et al., 2005). As a result, finding a non-destructive way to extract DNA from an organism for

DNA barcoding is important (Phillips & Simon, 1995).

Several studies have shown that barcoding can be done with DNA that is extracted in a

non-destructive manner (Phillips & Simon, 1995; Rholand et al., 2004; Pons, 2006; Gilbert,

Moore, Melchior, & Worobey, 2007; Rowley et al., 2007; Hunter et al., 2008). However, as

stated above, often times these experiments use harmful chemicals, such as phenol and

chloroform, require overnight incubation periods, and are not universal (Hunter et al., 2008;

Castalanelli et al., 2010). Additionally, in the majority of these experiments, DNA was not

extracted with the use of commercial kits. In fact, little research has been done on whether or not

DNA can be extracted in a non-destructive manner with a commercial kit (Lucentini et al., 2006;

Castalanelli et al., 2010). Extracting DNA in a non-destructive manner with the use of a


commercial kit, such as the Qiagen DNeasy Blood and Tissue Kit, could make non-destructive

DNA extraction quicker, more universal and more ideal for museum specimens, endangered

species, and voucher specimens (Chen et al., 2010). Thus, by using this mechanism, species, old

and new alike, could be barcoded efficiently.

DNA extraction of Spiders to Gain Barcodes

Since there are about 37,000 identified species of spiders and 40,000 suspected species of

spiders, barcoding each and every one of these species is a huge undertaking (Barrett & Hebert,

2005). DNA barcoding is ideal in spiders because of the complexity of their morphology. Often,

spider species cannot be differentiated unless the spider is an adult (Barrett & Hebert, 2005).

This poses a problem because spiders at any other life stage cannot be differentiated.

Additionally, adult spiders molt and grow as they age, which can make morphological

differentiation approaches nearly impossible (Barrett & Hebert, 2005). Lastly, in many species of

spiders, male and female spiders have differences in their morphologies that create challenges

when trying to determine their species (Barrett & Hebert, 2005). Thus, DNA barcoding makes

the most sense in helping differentiate spider species (Barrett & Hebert, 2005).

When trying to extract DNA from spiders, often time legs are removed to obtain DNA

usable for DNA barcoding (Casquet et al., 2012; Miller et al., 2013). This is problematic because

small spiders do not have enough DNA in their legs to produce a suitable DNA barcode. Thus, in

order to extract enough DNA to barcode a small spider, the whole voucher specimen is crushed

to obtain DNA (Castalanelli, et al., 2010). However, this technique is not ideal because it ruins

the entire voucher specimen (Rholand et al., 2004; Castalanelli et al., 2010). Yet, research shows

that DNA can be extracted from a small spider without removing its legs or crushing it

(Castalanelli et al., 2010). In fact, studies show that non-destructive, whole body DNA


extractions of small spiders do not do any significant damage to the spider, which makes non-

destructive DNA extraction techniques ideal for museum collections and voucher specimens

(Miller et al., 2013). However, the amount of DNA that is extracted can be prohibitively small

when derived from a non-destructive DNA extraction technique.

Recent studies have shown that DNA has been extracted from museum collections of

spiders, so fresh DNA is not necessary when attempting to barcode older spiders (Miller et al.,

2013). This is important because these results show that voucher specimens or museum

collections need not be harmed while extracting DNA. However, the tissue available for DNA

extraction expires faster in smaller spiders than in larger spiders, so finding a non-destructive,

commercial DNA extraction method that obtains enough DNA for barcoding is critical (Miller et

al., 2013).

One way to ensure that small quantities of DNA will work for downstream analyses is to

use a more specific primer. When extracting DNA that is usable for a DNA barcode sequence,

primer design is critical to ensure amplification of the correct fragment during PCR (Hajibabaei

et al., 2005; Casiraghi et al., 2010; Moulton et al., 2010). Using a primer that is more specific to

spiders could allow for complete sequencing of mitochondrial genomes when there is less DNA

available (Hajibabaei et al., 2005; Moulton et al., 2010). When barcoding spiders, several studies

have been successful using a standard forward primer, such as LCO1490 (5′-

GGTCAACAAATCATCATAAAGATATTGG-3′), and a spider-specific reverse primer, such as

chelicerate reverse 2 (5′-GGATGGCCAAAAAATCAAAATAAATG-3′; Barrett & Hebert,

2005; Miller et al., 2013). Thus, spiders are an ideal candidate for non-destructive DNA

extraction techniques using a commercial kit.


Methodology

Fresh Collections

Fresh collections of spiders (1-3 mm in size) were caught in Mitchell, South Dakota, by hand,

aspirator, or trap. After being caught, the samples were kept in 70% ethanol as a preservative

until they were worked on. There were 17 fresh specimen tested.

Voucher Collections

Small spiders (1-3 mm in size) were selected from the voucher collection of Dr. L. Brian Patrick

at Dakota Wesleyan University. These spiders were all four years old and were previously

collected in Fort Pierre National Grasslands in Stanley County, Jones County, or Lyman County

in South Dakota. These spiders were caught by a trap and identified by Dr. L. Brian Patrick. The

specimens were stored in closed vials in 70% ethanol. These spiders were stored in a cabinet and

were not exposed to light. There were 46 voucher specimen tested.

Figure 1. Stanley, Lyman, Jones and Davison County in South Dakota


Photographic Evidence

Before DNA extraction, each specimen was photographed under a Leica microscope. Specimens

were removed from the vials using pipettes. Then, specimens were stuck in petroleum jelly and

ethanol to prevent movement and to raise resolution. Next, the dorsal and ventral side of each

specimen was photographed. After DNA extraction, the specimens were photographed on their

dorsal and ventral sides again. If the legs of the spiders look more transparent in the after

photographs, it was an indication that DNA was extracted.

DNA Extraction

DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, 2006). For

destructive samples, all protocols were followed except: instead of 200 µL, only 100 µL of

Buffer AE were added after the last extraction step to increase the final DNA concentration in

the elute. For non-destructive samples, all protocols were followed except: the whole specimen

was incubated rather than grinding up the specimen, the sample was allowed to incubate for 4

hours instead of 3 hours, the sample was mixed every hour using a pipette rather than a vortexer,

and, after incubation, the sample was pulled out of the test tube before vortexing instead of

vortexing the specimen with the sample. In order to avoid destroying small specimens, samples

were placed into their microcentrifuge tubes via a pipette. This ensured that DNA was extracted

in a non-destructive manner using a commercial kit. The amount of DNA extracted was

measured using a Qubit Fluorometer, which can detect amounts of DNA equal to or greater than

25 ng/µL.

PCR

PCR was performed using the Takara Taq reagents. PCR was done on 25 µL reactions, and all

Takara reagents and DNA template were calculated accordingly. As such, 421.875 µL of water,


62.5 µL of Taq buffer, 50 µL of deoxyribonucleotides, 12.5 µL of each primer, and 3.125 µL of

Taq were mixed into a stock solution that performed 25 reactions each with a total volume of 25

µL. For each sample, 2.5 µL of DNA template were added to the PCR tube. The primers used

were LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) (Folmer, BlacK, Hoeh, Lutz,

& Vrijenhoek 1994) and Chelicerate Reverse 2 (5’-CCTCCTCCTGAAGGGTCAAAAAATGA-

3’) (Barrett & Hebert 2005), which have been shown to work well on spiders. PCR was

performed with initial denaturing at 94°C for 60 seconds. PCR then went through 32 cycles of

denaturing at 94°C for 45 seconds, annealing at 48°C for 45 seconds, and extension at 72°C for

30 seconds. The final extension was done at 72°C for 300 seconds. When PCR was complete,

samples were refrigerated at -20°C. PCR sequencing was done by the CORE sequencing facility

at Black Hills State University. Barcode sequences will be uploaded to GenBank. Samples C49-

C60, C182-C183, C194-C204, C206, C208-C209, and C211-C212 were ran through another

PCR machine after it was discovered that the first PCR machine broke.


Results

Fresh Collections

Of the 17 fresh samples, detectable DNA was extracted in a non-destructive manner using a

commercial kit in all 17 samples (Table 1). This means that at least 25 ng/µL of DNA was

present in each sample after DNA extraction. The average DNA concentration for the 17 samples

was 217.59 ng/µL. After removing an outlier, the average DNA concentration of the 17 samples

was 156.81 ng/µL. In addition, of these 17 samples, a PCR result was obtained from nine of

them. This means that in about 53% of the samples, the DNA extracted in a non-destructive

manner using a commercial kit allowed for DNA amplification and a DNA barcode could be

obtained. The average DNA concentration of the samples that obtained a PCR result was 330.28

ng/µL. For sample C267, 290 ng/µL of DNA was extracted. A change can be seen between the

before and after photographs in the legs of spider, especially at the joints (Figure 1 and Figure 2).

For all samples, two bands on the PCR gel pictures indicates that a PCR result was obtained, and

a single, small band represents a failed reaction (Picture 1). An arrow at the top of the gel picture

indicates the placement of the target amplification product in non-destructive samples.


Table 1. DNA Concentration and PCR Result of Fresh Collection Samples

Sample Number DNA [C] ng/ µL PCR Result Obtained

C194 276 Yes

C195 325 Yes

C196 80.5 No

C197 288 Yes

C198 179 Yes

C199 134 Yes

C200 194 Yes

C201 37.5 No

C202 80 No

C203 44.5 No

C204 1190 Yes

C206 60.5 Yes

C208 27.5 No

C209 130 No

C211 36.5 No

C212 326 Yes

C267 290 No


Picture 1. PCR Results of Fresh Collections C194-C204, C206, C208-C209, and C211-C212


Figure 2. Sample C267 before DNA extraction

Figure 3. Sample C267 after DNA Extraction


Voucher Collections

Of the 46 samples voucher samples, detectable DNA (greater than 25 ng/µL of DNA per sample)

was extracted in a non-destructive manner using a commercial kit in 16 samples (Table 2). The

average DNA concentration of these 16 samples was 162.19 ng/µL. After removing an outlier,

the average DNA concentration of the samples with detectable DNA was 114.33 ng/µL. Of these

16 samples, a PCR result was obtained in 4 samples. The average DNA concentration of those 4

samples was 375.13 ng/µL. Additionally, 4 samples that had no detectable DNA (less than 25

ng/ L of DNA per sample) obtained a PCR result (Table 3). This means that about in about 17%

of the samples, the DNA extracted in a non-destructive manner using a commercial kit allowed

for DNA amplification and a DNA barcode could be obtained.


Table 2. Voucher Collection Samples that had Detectable DNA Concentrations


C51 255 Yes

C52 234 No

C53 49.5 Yes

C151 91 No

C158 31 No

C160 35.5 No

C162 207 No

C163 32 No

C169 240 No

C171 50.5 No

C255 58 No

C261 30.5 No

C264 36 No

C268 49 No

C182 880 Yes

C183 316 Yes


Table 3. Voucher Collection Samples that had no Detectable DNA and Yielded a PCR Result


C50 <25 Yes

C54 <25 Yes

C59 <25 Yes

C253 <25 Yes


Figure 3. Sample C261 before DNA extraction

Figure 4. Sample C261 after DNA extraction


Discussion

The results of this study have many implications. First, since all the fresh collection

samples had detectable DNA and over fifty percent of the fresh collections yielded a PCR result,

it can be concluded that a non-destructive DNA extraction method using a commercial kit works

reasonably well on fresh specimens. However, for the voucher collections, only four samples out

of 46 had detectable DNA concentrations and only eight samples out of 46 yielded PCR results.

Thus, it can be concluded that non-destructive DNA extraction techniques using a commercial

kit are not as efficient when working with older or preserved samples.

On average, fresh collections that had detectable DNA concentrations had higher

amounts of DNA than voucher collections that had detectable DNA concentrations. The fresh

collections had an average of 55.4 ng/µL more DNA per sample than the voucher collections.

Additionally, a higher average DNA concentration was needed to obtain a PCR result in the

voucher collections. It took, on average, 44.85 ng/µL more DNA to obtain a PCR result in

voucher collections. This may have something to do with the quality of DNA obtained in

voucher collections.

Because this technique worked fairly well with fresh samples and did not work well with

voucher specimens, it can be determined that this technique should only be used on fresh

collections. This technique could be helpful in instances when a fresh sample has been caught

and not yet identified. Knowing this information, researchers could make sure that they extract

DNA in a non-destructive manner within a certain time period of catching a specimen. This

could allow researchers to extract DNA in a non-destructive manner using a commercial kit,

perform PCR, and enter sequences into a database, all while preserving the specimen as a

voucher.


Conclusions

One limitation of this study was the age of the voucher collections. Since all of the

voucher collection samples in this study were over three years old, an interesting extension of

this project could be to see how long a voucher specimen can sit before efficacy of the technique

drops significantly. One way to test this would be to test voucher specimens that had been

collected six months previously, a year previously, a year and a half previously, two years

previously, and 3 years previously to see if DNA extraction decreases significantly at any of

these points.

Another limitation of this study was how long some of the spiders were in the traps after

collection. Traps were set up in two week increments in Stanley, Jones and Lyman County in

South Dakota. As a result, some spiders could have spent up to two weeks in the traps. The

sooner a spider is drowned and preserved, the easier it is to extract DNA from it. If a spider dies

and is not placed in 70% ethanol immediately, it can lose DNA as its proteins denature. Thus, the

length of time that the spiders spent in the traps could have affected the amount of DNA

extracted.

The way some of the voucher specimens were stored also could have led to less DNA

extraction. On accident, three years previously, several samples had been stored in 70% ethanol

that had been diluted with tap water rather than distilled water. These samples developed a

residue on them when removed from their storage vials. This residue could have prevented DNA

extraction or lead to degradation of DNA.

The last limitation of this study was that all of the fresh collections were caught in

Davison County, South Dakota, while all of the voucher collections were caught in Stanley,

Lyman and Jones Counties in South Dakota. To remedy this problem, fresh collections and


voucher collections from all four counties could be tested. If the same results were achieved, it

would prove that there is a difference between fresh collections and voucher collections and not

between geographical areas.

Additionally, some changes to the procedure could change the results of this experiment.

For instance, before PCR, rather than adding 421.875 µL of water to the pre-made stock solution

and then adding 22.5 µL of the stock solution with 2.5 µL of DNA, 16.88 µL of water and the

5.62 µL of the pre-made stock solution could be added to each individual DNA extraction

sample. This would allow for less dilution of the stock solution and ensure that each sample of

DNA was given an adequate amount of stock solution.

The purpose of this quantitative research was to extract sequence-able DNA, known as a

DNA barcode, from small spiders using non-destructive DNA extraction techniques and the

Qiagen DNeasy kit (Qiagen, 2006). The DNA extracted from these spiders could be entered into

a national database, such as GenBank or CBOL, to identify existing species and confirm new

species. It is important to explore Non-destructive DNA extraction techniques because they

allow for preservation of the voucher specimen. Though this method worked better in fresh

collections than voucher collections, it could still be used to preserve voucher specimens as long

as DNA is extracted within a timely matter. Using a commercial DNA extraction kit could make

the process of extracting DNA from small voucher specimens more regulated, and thus more

universal. The methods described in this study were as simplified as possible to ensure that other

labs could replicate the technique.

This study was significant in that it tested the efficacy of non-destructive DNA extraction

methods using a commercial kit. Though the results were not as expected, they are still

noteworthy. From this study, it was found that DNA can be extracted in a non-destructive


manner using a commercial kit if the specimen is fresh enough. Knowing this, researchers can be

sure to extract DNA from voucher specimens as soon as possible. This guarantees that DNA can

be extracted and the specimen can be saved as a voucher. Then, the DNA barcodes of those

specimens can be logged into GenBank or CBOL. For this reason, it is important to understand

and expand on the results from this experiment.


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